Biofuel is defined as solid, liquid or gaseous fuel derived from relatively recently dead biological material (and is thereby distinguished from fossil fuels, which are derived from long dead biological material). Theoretically, biofuels can be produced from any (biological) carbon source; although, the most common sources are photosynthetic plants. Various plants and plant-derived materials are used for biofuel manufacturing. Agrofuels are biofuels which are produced from specific crops, rather than from waste processes.
There are two common strategies of producing liquid and gaseous agrofuels. One is to grow crops high in sugar (sugar cane, sugar beet, and sweet sorghum) or starch (corn/maize), and then use yeast fermentation to produce ethyl alcohol (ethanol). The second is to grow plants that contain high amounts of vegetable oil, such as oil palm, soybean, algae, jatropha, or pongamia pinnata. When these oils are heated, their viscosity is reduced, and they can be burned directly in a diesel engine, or they can be chemically processed to produce fuels such as biodiesel. Wood and its byproducts can also be converted into biofuels such as methanol or ethanol fuel.
Algae can be used as a source for several types of biofuels with the most studied one being biodiesel. Algae are fast-growing organisms with naturally high oil content that can be extracted year-round. Algae can be grown in non-arable areas and thus do not compete with food crops on land. Algae provide the most promising long-term solution for sustainable biofuels production with only a minimal cropping area required to supplement a large fuel supply. Interest in algae is recently growing as a potential feedstock for biodiesel production with the emerging concern about global warming that is associated with the increased usage of fossil fuels.
Algal biotechnology has made considerable advances in recent years including development of methods for genetic transformation and sequencing the genomes of several algal species and significant efforts have been made over the last 15 years to increase the oil yield in algae. Oil content in algae is known to be influenced by several environmental and nutritional factors such as nitrogen starvation and strong light. Although much has been learned about genetic control of oil synthesis in algae over that time, researchers have still not been able to significantly increase the oil yield of algae by genetic engineering.
MicroRNAs are 17-24 nucleotide long, endogenous RNAs that regulate gene expression in plants and animals. MicroRNAs are processed from stem-loop regions of long primary transcripts and are loaded into silencing complexes, where in plants they generally direct cleavage of complementary mRNAs. MicroRNAs play crucial roles at each major stage of plant development, typically at the cores of gene regulatory networks, usually targeting genes that are themselves regulators thus affecting the abundance or stability of numerous genes at once.
Recently, microRNAs have been discovered in the unicellular model alga Chlamydomonas reinhardtii and preliminary evidence indicates they play crucial roles in algal development (Molnar et al., 2007, Nature 447:1126-1130).
So far, plant microRNAs that target genes involved in abiotic and biotic stress responses, hormone signaling and metabolism and the microRNA machinery itself have been identified (Jones-Rhoades et al., 2006, Annu Rev Plant Biol 57:19-53).
A commonly-used approach in identifying the function of novel genes is through a loss-of-function mutant screening. In many cases, functional redundancy exists between genes that are members of the same family. When this happens, a mutation in one gene member might have a reduced or even non-existing phenotype and the mutant lines might not be identified in the screening.
Using microRNAs, multiple members of the same gene family can be silenced simultaneously, giving rise to much more intense phenotypes. This approach is also superior to RNA interference (RNAi) techniques where typically 100-800 bp fragments of the gene of interest form a fold-back structure when expressed. These long fold-back RNAs form many different small RNAs and prediction of small RNA targets other then the perfectly complementary intended targets are therefore very hard. MicroRNAs in contrast, are produced from precursors, which are normally processed such that preferentially one single stable small RNA is generated, thus significantly minimizing the “off-target” effect.
A second approach of functional screening is through overexpression of genes of interest and testing for their phenotypes. In many cases, attempting to overexpress a gene which is under microRNA regulation results in no significant increase in the gene's transcript. This can be overcome either by expressing a microRNA-resistant version of the gene or by downregulating the microRNA itself.
Attempts to increase oil yield in algae through standard molecular biology techniques was so far only marginally successful. In recent years, major effort was focused on studying the Acetyl-CoA carboxylase (ACCase) enzyme, which is considered to regulate a key step in fatty acid biosynthesis. While efforts focused on genetic manipulation to increase the activity of ACCase have been going on for at least 15 years, the research has not yet reached the stage of actually being able to substantially increase the net oil yield from algae.
There is an unmet need to efficiently introduce significant changes in plants traits such as oil content.